In diseases of the nervous system such as Parkinson’s disease (PD) and Lou Gehrig’s disease very specific groups of nerve cells die. At least in the case of PD, the surgical methods exist for the implantation of new cells into the area of the brain where the nerve cells are dying. However, since fetal brain cells are almost impossible to obtain, an alternative must be found. A viable and untested potential source of brain neurons are human embryonic stem (ES) cells. In order to get these cells to function in the brain, it is mandatory that the ES cells be converted to nerve cells before they can be surgically implanted. In our past work with rat and mouse stem cells we have been able to identify and purify factors that are made by nerve precursor cells that cause stem cells to become neurons. In addition, we have a very large potential source of these types of factors that is unique to our laboratory in the form of embryonic cell lines from the brain. Therefore, the goal of this proposal is to identify new factors that convert human ES cells to specific types of neurons. Because we have previously identified nerve differentiation factors from our cell lines, we are certain that there are additional molecules to be discovered. These growth and differentiation factors will be of enormous help in meeting the technical requirements for successfully using human stem cells for the treatment of chronic neurological diseases.
Statement of Benefit to California:
Our work will benefit the State in a number of ways. 1) There could be a tremendous health benefit for individuals with diseases of the brain such as Parkinson’s and Lou Gehrig’s diseases as well as damage due to stroke or trauma. 2) Support for this work will provide current employment within the State and help educate scientists in the stem cell field. 3) The advancement of work on novel growth factors will require the collaboration with commercial (for profit) companies. Most of early stage preclinical development is done in small biotech companies, many of which are within the State. Therefore, there is an economic benefit to the State as well as a health benefit to the State and the world if some of the most debilitating human diseases could be cured.
This proposal is focused on the identification of growth factors that promote neural differentiation and proliferation of human embryonic stem cells (hESCs). The applicant has a unique collection of over 100 cell lines derived from rat embryo brains and proposes to screen conditioned media from these lines for the ability to promote differentiation of hESCs into neuronal phenotypes. Mass spectrometry will then be used to identify any active secreted factors from these conditioned media. In parallel, using an Oct-4-GFP knock-in hESC line, the applicant proposes to screen the same conditioned media for the ability to promote proliferation, and again identify the proteins responsible by mass spectrometry.
The reviewers were enthusiastic about the importance of developing neuronal differentiation protocols for hESCs and were very supportive of the team assembled to execute this project. However, they commented that weaknesses in experimental design and a low commitment of personnel raised questions about the feasibility of the project.
Reviewers agreed that the proposal addresses an important area of stem cell research and has the potential for high impact in the field. There is a pressing need for the identification of factors that promote the differentiation of hESCs into highly enriched populations of specific cell types, and existing protocols for neural differentiation are often lacking in efficacy and reproducibility. In addition, the second goal of identifying factors that promote the self-renewal and propagation of hESCs was also considered important. Two reviewers thought the application would be much stronger if it proposed to examine differentiation into specific neuronal cell types, but after discussion it was agreed that this objective was beyond the scope of a 2-year proposal.
The major risk in this project is whether the relevant biological activities can be identified using conditioned medium, and whether the purification strategies are robust enough to be able to identify the relevant molecules. One reviewer commented that the applicant would only be able to identify active factors involved in differentiation and maintenance of pluripotency if they are proteins, and non-protein factors are certainly going to be important. Another noted that although the collection of the conditioned medium would then be done in serum-free medium, cell lines would be propagated in media containing serum and all subsequent steps would use the same flasks. From experience, this reviewer warned that even after washing there would still be considerable contamination by serum proteins in higher abundance than many secreted growth factors. Serum contamination was not addressed in the application and would make isolation of active factors with mass spectrometry more difficult. One reviewer was unsure about how the applicant would prioritize which individual proteins from the mass spectrometry screen to take further. It is likely that the researchers would end up with fractions containing several if not many different proteins and would need a selection process to prioritize which of these to clone and test, but there is little mention of the strategy in the design and methods. Finally, reviewers were concerned about the lack of detail provided on how neural differentiation would be assessed, and felt that the definition of a neuronal phenotype was not well articulated.
Reviewers also expressed disappointment that no high-throughput methods were proposed and commented that much of this project could have been carried out ten years ago. The use of the Oct4-GFP knock-in cell line was considered a good strategy and the isolation of factors that promote hES cell pluripotency would be useful, but attempts to purify such factors from conditioned medium of mouse or human fibroblasts have proved unfruitful in other labs, despite years of work. It’s unclear that this research team has any competitive advantage in this area, and no novel strategies were proposed in the application. Reviewers also mentioned the proposal’s demanding criteria for success: identification of two factors capable of 100% irreversible differentiation of hESCs into neurons or 100% continuous cell proliferation by the end of year one. While reviewers praised the applicant’s willingness to forego a second year of funding if unsuccessful, this also contributed to the reviewers’ concern that the project was not feasible as written.
The reviewers agreed that the applicant has outstanding resources to tackle such an ambitious project. The applicant has extensive neurobiology and neural stem cell experience and has identified several novel growth factors – the applicant is therefore uniquely qualified to lead the proposed research. The applicant’s institution is well equipped with the facilities and staff required to undertake the proposed experiments, including experience in protein chemistry and mass spectroscopy. In addition, one of the listed collaborators is a highly experienced stem cell biologist. Reviewers’ main concern was that the 10% effort allocated to the project by the primary investigators and the dedication of only one full-time trainee to the project would not be enough to carry this project forward successfully: They would have preferred to see a greater commitment. The budget seems appropriate and justified.
Overall, this is a highly ambitious proposal that, if successful, would significantly advance the field of stem cell biology. However, the reviewers raised several serious concerns about the feasibility of the research plan as written, and felt that the proposal did not take advantage of the most modern methodologies.